Hyaluronan (HA), which is a ubiquitous extracellular matrix component in vertebrates, is a powerful modulator of cell behavior, e.g. during cell migration, development, cancer, wound healing and angiogenesis. Accumulating evidence suggests that alterations in the normal synthesis and degradation of HA can be critical in the pathogenesis of diseases such as arthritis and cancer. The long-term hypothesis guiding this project is that HA synthesis must be tightly controlled in order to achieve normal homeostasis and health. The overall project has contained two sub-projects that have investigated HA synthesis (mediated by HA synthases, which we were the first group to clone and then to purify) and HA turnover (mediated by the HA Receptor for Endocytosis). Because these sub-projects now have a molecular basis and have greatly expanded, they are being split into two separate R01 projects. The current project will continue to study the streptococcal and human HA synthases (HASs). However, the HAS field is presently stalled for two reasons: no one has been able to purify an active native human HAS, or to assess the amount or activity of one individual HAS isoform in the presence of the other two. Consequently, our strategy to advance the field is to use the streptococcal HASs as a model to understand the functions of the human HASs. Our specific hypothesis is t hat structure-function analyses of the streptococcal HASs will allow us to understand how the human HASs work and how HA synthesis is regulated. We will continue efforts to understand the streptococcal HASs, hut also use what we learn to study the human HASs. We will employ techniques in biochemistry, molecular biology and cell biology to address multiple hypotheses in the following specific aims. 1) To characterize the novel HA-UDP linkage at the reducing end of newly synthesized HA. 2) To determine the role of chitin-UDP oligomers in HA synthesis by HAS. 3) To identify the unique phospholipids that activate seHAS and test their effects on human HASs. 4) To analyze structure-function relationships in the streptococcal HASs. 5) To determine the organization and motion of seHAS domains using EPR spectroscopy. Results from this project could lead to the first investigations of the roles of human HAS proteins in various pathologies and diseases and could also lead to the development of new drugs for streptococcal diseases.